Sex and estrous cycle effects on stress-enhanced fear learning in Long-Evans rats

Abstract

Individuals diagnosed with post-traumatic stress disorder (PTSD) are hyperresponsive to otherwise ordinary stimuli (e.g., loud noises or certain smells) long after a traumatic experience. At a preclinical level, this persistent effect of trauma is captured in stress-enhanced fear learning (SEFL), in which a stressful experience in one context causes a persistent increase in fear conditioning with a mild stressor in a second context. Here, we characterized multiple behaviors (including freezing, rearing, darting, and jumping) in male and female Long-Evans rats during SEFL. Rats received a battery of foot shocks in one context followed by a mild stressor (single foot shock) in a second context. We found that males and females with a history of multiple shocks in one context had higher freezing levels during the SEFL test, relative to no shock controls, and that this SEFL effect persisted to a second test 30 days later. The dominant response was freezing in males and females, with no reliable sex differences at any stage of the SEFL procedure, but females also showed some unique escape-like behaviors on the days of trauma and mild stress exposure. In addition, females in the proestrus and estrus phases of the estrous cycle during the initial shock exposure showed increased SEFL relative to females in the metestrus and diestrus phases. These findings suggest that males and females show similar SEFL effects with freezing behavior, but that ovarian cycle phase at the time of trauma may alter the strength of SEFL in females.

Figure 1.

Experimental timeline. The stress-enhanced fear learning (SEFL) procedure, used in both Experiment 1 and Experiment 2, included the following: 2 days of contexts pre-exposure, 1 day of foot shock exposure (varying by group: 0, 1, 4, or 15 foot shocks in Experiment 1; 0 or 15 foot shocks in Experiment 2), a single foot shock in a different context on the following day, and two SEFL tests conducted at 24 hours and 30 days later.

Figure 2.

Behavioral profiles of male and female rats during the battery of foot shocks in Context A in Experiment 1. Data are presented as mean or mean + SEM percent time. Eight different behaviors were quantified during the foot shock sessions, with session duration varying by shock condition: 5 min (1 shock), 24 min (4 shocks), or 90 min (0 and 15 shocks). (A–H) Mean percent time spent engaging in each behavior for the 0-shock (A,E), 1-shock (B,F), 4-shock (C,G), or 15-shock (D,H) groups. No significant sex differences were observed (males: A–D; females: E–H). Collapsing data across sexes revealed significant differences between shock groups: (I) Freezing was significantly greater in the 4- and 15-shock groups compared to 0- and 1-shock groups. Individual data points are shown, with open circles representing males and solid triangles representing females. (J) In the 15-shock group, freezing behavior significantly increased as early as the 32 sec window preceding the second shock and remained elevated across most of the shocks, relative to matched timepoints in the 0-shock group. This effect was also independent of sex, with males represented by a dotted black line and females a solid black line. (K) Freezing response was primarily observed during the pre-shock timepoint rather than immediately post-shock. (L–Q) Other nonfreezing behaviors showed differential engagement before and after shock. In the 15-shock group, general movement (M) and rearing (N) increased during post-shock timepoints. Although not statistically significant, darting was observed exclusively post-shock in a few females. (*) P < 0.05.

Figure 3.

Freezing response in Context B. Data are presented as mean + SEM percent time in Experiment 1. Sexes are collapsed due to the absence of significant sex differences but are represented by individual data points (open circles = males; solid triangles = females). (A) During the 5 min single shock session in Context B, rats in the 15-shock group exhibited significantly greater freezing behavior compared to the 0-shock controls. Regardless of shock group, all rats displayed a significant increase in freezing behavior at the post-shock timepoint (32 sec window, four samplings) compared to the pre-shock timepoint (32 sec window, four samplings). (B) When re-exposed to Context B during the 12 min stress-enhanced fear learning (SEFL) tests, Test 1 (left panel) conducted 24 h later and Test 2 (right panel) conducted 30 days later, the 4- and 15-shock groups exhibited heightened freezing behavior relative to 0- and 1-shock groups. These shock group differences persisted despite an overall reduction in freezing behavior from Test 1 to 2. (*) P < 0.05.

Figure 4.

Female behavioral profiles during the battery of foot shocks in Context A across metestrus-diestrus (M-D) and proestrus-estrus (P-E) phases of the estrous cycle. Data are presented as mean or mean + SEM percent time. Eight behaviors were quantified during the 90 min sessions involving either 15 foot shocks (“trauma” experience) or no shocks. Panels A and C show the 0-shock control groups, while panels B and D show the 15-shock groups. Significant estrous cycle phase differences were observed. Among the 0-shock controls (A,C), females in the M-D phases (A) spent significantly more time moving but less time grooming compared to females in the P-E phases (C). In the 15-shock groups (B,D), females in the M-D phases (B) spent less time moving and more time immobile in rearing position compared to P-E females (D). Shock group differences (0 vs. 15 shocks) were consistent with patterns observed in Experiment 1, except for rearing and darting behavior. P-E rats exposed to 15 shocks engaged in less rearing behavior than their 0-shock counterparts (also see I). Although darting was also rare in Experiment 2, it occurred predominantly in P-E phase rats subjected to 15 shocks compared to P-E 0-shock controls (also see L), differences not evident in M-D rats. (E) Independent of estrous cycle phase (black dotted line = P-E; black solid line = M-D), a single foot shock elicited a significantly enhanced freezing response in the shock groups, compared to controls. (F) Similar to Experiment 1, significantly more freezing behavior was observed at the pre-shock timepoint compared to post-shock. Panels G–L depict other behaviors, highlighting differential engagement before and after shock. Within the 15-shock group, a notable increase was observed in (H) general movement, (I) rearing, and (K) jumping during post-shock timepoints. The only significant main effects or interactions involving cycle phase were observed for moving and immobility in a rearing position: 15-shock P-E rats (see H, open circles) exhibited significantly more movement overall compared to 15-shock M-D rats, while 15-shock M-D rats engaged more in immobility in a rearing position (see G, solid triangles). (*) P < 0.05.

Figure 5.

Freezing response in Context B in Experiment 2. Data are presented as mean + SEM percent time. The x-axes indicate the estrous cycle phase classification at the time of trauma or no trauma exposure in Context A on experimental Day 3 (A,B), or at the time of mild stress exposure on Day 4 (C,D). Individual data points represent the estrous phases: M-D = solid triangles, P-E = open circles. (A) During the 5 min single shock session in Context B, rats in the 15-shock group exhibited significantly greater freezing than those in the 0-shock control group. Regardless of shock group, freezing increased from the pre- to post-shock timepoint (each consisting of 32 sec windows averaged across four samplings). However, an interaction between Day 3 estrous cycle phase and timepoint revealed that P-E rats (open circles) deviate from this pattern, showing no significant change in freezing behavior from pre- to post-shock. (B) During the 12 min SEFL Test 1 (left panel) and Test 2 (right panel), both conducted in Context B, the 15-shock groups showed elevated freezing compared to 0-shock controls. These differences persisted despite an overall reduction in freezing behavior from Test 1 to Test 2. An interaction between cycle phase, shock group, and test day indicated that this reduction in the 15-shock group was driven by P-E females (open circles). Additionally, a cycle phase difference (M-D vs. P-E) was apparent only at Test 1 (24 h test day), with P-E rats freezing significantly more than M-D within the shock group. (C) Reclassifying cycle phase based on Day 4 (the day of single shock) revealed similar main effects as in A. However, a significant cycle phase, group, and timepoint interaction emerged: P-E rats in the 15-shock group showed a significant increase from pre- to post-shock, whereas M-D rats did not, regardless of shock history. (D) When using Day 4 mild stress classifications, no effect of cycle phase was observed during the SEFL tests. Rats in the 15-shock group froze more than controls across both test days, and freezing significantly decreased from Test 1 to Test 2. (*) P < 0.05.